The present invention relates to compounds and methods for the prevention and treatment of microbial infection of a mammalian host through the administration of substrates for transglutaminases or antibodies against such substrates that inhibit the transglutaminase-mediated interaction of the microorganism with the mammalian host. These compounds and methods may be used preferably in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised or immunosuppressed humans, for example, those having AIDS or undergoing transplantation or anti-cancer therapy.
Whether pathogenic or opportunistic, microorganisms have evolved numerous mechanisms to facilitate their establishment and proliferation in mammalian hosts. During initial infection, the interaction of a microorganism with its mammalian host can include attachment or adhesion to the host cell surface, invasion of host cells, and elaboration of toxins, for example. In certain instances, this interaction can be nonspecific. In others, such microbial interaction involves the specific binding of the microorganism to a particular receptor or receptor complex expressed on the host cell surface. In turn, the binding event can trigger changes in the microorganism and/or the mammalian host cell, leading to the progression of infection.
The host cell functions of molecules involved in certain microbial interaction are unknown in some cases and known in others. Mammalian transglutaminases are examples of those in the latter category for which the molecular mechanism of action and/or role in host cell growth or development has been elucidated. In general, transglutaminases are enzymes that catalyze intermolecular crosslinks by the formation of highly stable isodipeptide bonds between the xcex3-carbonyl group of glutamine and the xcex5-amino group of lysine residues, which are resistant to proteases, sodium dodecyl sulfate and heat. Epithelial cell transglutaminases are important for the formation of cornified envelopes of mature squamous epithelial cells.
Only recently have investigators shown that certain microorganisms may express proteins capable of acting as substrates for, and thus interact with, mammalian transglutaminases. One example is hyphal wall protein 1 (Hwp1), which is expressed on hyphal surfaces of the pathogenic fungus, Candida albicans. Hwp1 consists of an N-terminal proline and glutamine-rich repetitive amino acid sequence that is exposed on the hyphal surface, and a cell wall-anchored serine and threonine-rich C-terminus. The composition of the N-terminal amino acid repeats is reminiscent of mammalian transglutaminase substrates. It is now known that Hwp1 can serve as a substrate in transglutaminase-mediated cross-linking reactions.
Candida is an ubiquitous yeast recognized as the causative agent of candidiasis (Candida mycosis). At least 90% of the disorders are caused by the species C. albicans, which is an opportunistic yeast able only to elicit mild superficial infections in normal individuals. Fungal infections associated with severe infections of the mucous membrane and with invasive infections of individual organs are observed ever more frequently as a result of the increasing number of patients with immune defense weakness, e.g., patients with acquired immunodeficiency syndrome (AIDS) or patients undergoing immunosuppressive therapy.
If left untreated, such systemic infections frequently lead to the death of the patients. At present, the treatment for invasive infections is based on relatively few antimycotics, such as amphotericin B and flucytosine, or the azole derivatives fluconazole and itraconazole. These antimycotics cause serious, sometimes different, side effects, such as renal insufficiency, hypocalcemia and anemia, as well as unpleasant constitutional symptoms such as fever, shivering and low blood pressure.
For this reason, doctors and clinicians are interested, for achieving direct and effective therapy, in having available diagnostic procedures permitting the earliest possible identification of the fungal pathogens. Conventional methods of diagnosis are based on the in vitro cultivation of the pathogens and the identification of the fungal species by means of morphological, physiological and biochemical methods. The culturing of C. albicans from blood is frequently very difficult and unreliable. Although C. albicans can be cultured from the mouths of normal persons, the progression to mucosal candidiasis is characterized by a shift in the microbial flora that includes an increase in the number of fungi in saliva, followed ultimately by invasion and inflammation of the gastrointestinal mucosa by C. albicans. The clinical presentations are pseudomembranous or erythematous lesions in the oral cavity and/or esophagus.
Oropharyngeal and esophageal candidiasis are among the most frequent opportunistic fungal infections observed in human immunodeficiency virus positive (HIV+) and AIDS patients, occurring in the majority of patients. The pathogenesis is complex and is thought to involve multiple host factors that include loss of cell mediated immunity and altered phagocytic cell activity. The current status of the AIDS epidemic is one of increasing numbers of individuals infected and no cure. Many infected individuals may live for a long time with HIV in an essentially permanent immunocompromised state. Because of the loss of the cellular component of the immune system, AIDS patients are susceptible to invasion of submucosal tissue by C. albicans. The frequency of candidal infections may also be a result of the prophylactic use of antibacterial drugs used in AIDS patients to minimize other opportunistic infections. Candidal infections increase in severity and recur more frequently as the immunodeficiency progresses.
While treatment with antifungal drugs can be effective, the increasing frequency of resistant strains of C. albicans, and the systemic side effects of the drugs prompts exploration of novel strategies to interrupt the sequence of events leading to disease and to expand the repertoire of antifungal drugs. An antifungal strategy based on biological interactions between C. albicans and the oral mucosa would be of great benefit to those with such fungal infections, e.g., patients with long-term immunodeficiencies.
Relevant features of C. albicans, the most frequent cause of oral candidiasis in HIV infected patients, are persistence in the gastrointestinal mucosa and invasiveness in the presence of diminished host defenses. Although C. albicans is sensitive to antifungal drugs, treatment over long periods of time are required, and isolates from HIV infected patients may be more resistant than other isolates. In addition to HIV infected patients, oral candidiasis occurs in patients with leukemia or other cancers, as well as in patients with other underlying diseases. Candidiasis in denture wearers, or denture stomatities, is the commonest of all C. albicans associated diseases. Indeed, new approaches towards preventing or managing oral candidiasis are needed.
A feature of C. albicans growth that is correlated with pathogenicity in the oral cavity is the ability to transform from budding to filament-extending growth. Filamentous forms adhere more readily to buccal epithelial cells than budding yeasts, and histologically are a prominent feature of invasion of the mucosa. Knowledge of the molecular events that transform C. albicans to the pathogenic filamentous form as well as detailed investigations of the hyphal surface at the molecular level are necessary for understanding the pathogenesis of oral candidiasis.
In mucosal and systemic disease, C. albicans exists as a polymorphic set of growth forms termed yeasts, pseudohyphae and true hyphae. In mucosal disease, filamentous forms, particularly true hyphae, invade the keratinized layer of differentiated, stratified squamous epithelium. True hyphae are septate, cylindrical structures with parallel sides that are formed by extension of germ tubes which emerge from yeasts in appropriate environmental conditions.
In the oral mucosa, Hwp1 adherence may help C. albicans resist the mechanical forces that clear the oral mucosa, enhancing colonization. Hwp1-mediated stabilized adhesion may induce accelerated maturation of epithelial cells, partially explaining the association of candidiasis with increased turnover of basal keratinocytes. Alternatively, Hwp1 may be important for interacting with host transglutaminases other than those associated with epithelial cells. Hwp1 may also be important for the spatial expression of other pathogenically-important proteins on the germ tube surface.
A valuable contribution to the art therefore would be compounds and methods for the prevention and treatment of microbial infection of a mammalian host through the administration of substrates for transglutaminases or antibodies against such substrates that inhibit the transglutaminase-mediated interaction of the microorganism with the mammalian host. These compounds and methods may be used preferably in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised humans having AIDS.
Accordingly, an objective of the present invention includes compounds and methods for the prevention and treatment of a microbial infection of a mammalian host through the administration of substrates for transglutaminases or antibodies against such substrates that inhibit the transglutaminase-mediated interaction of the microorganism with the mammalian host.
Another objective pertains to compounds and methods for the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised humans having AIDS. Yet another objective relates to the identification of the absence or presence of microbial infection and the site(s) of such infection. A further objective is a diagnostic kit for such identification. One other objective of the present invention is the prevention and treatment of a microbial infection of a mammalian host through gene therapy, whereby host cells, for example, are engineered to express substrates for transglutaminases that inhibit the transglutaminase-mediated interaction of the microorganism with the mammalian host. These and other objectives are achieved through the following preferred embodiments.
One aspect of the invention is a purified polypeptide comprising the amino acid sequence of SEQ. ID NO. 1, wherein said polypeptide is capable of acting as a substrate for mammalian transglutaminases. Another aspect is an isolated DNA molecule encoding the polypeptide having the amino acid sequence of SEQ. ID NO. 1, and an isolated DNA molecule comprising the nucleotide sequence encoding the polypeptide of SEQ. ID NO. 1. A further aspect is a nucleic acid capable of hybridizing under high stringency conditions to the DNA molecule of an isolated DNA molecule comprising the nucleotide sequence encoding the polypeptide of SEQ. ID NO. 1. In addition, an aspect of the invention is a vector comprising DNA encoding the polypeptide of SEQ. ID NO. 1, a host cell transformed with that vector, and that transformed host cell which produces a protein capable of acting as a substrate for mammalian transglutaminases.
An aspect of the present invention is an isolated antibody against the polypeptide comprising the amino acid sequence of SEQ. ID NO. 1 (or an antigenic portion thereof), wherein said polypeptide (or an antigenic portion thereof) is capable of acting as a substrate for mammalian transglutaminases. In a preferred embodiment, the antibody is a monoclonal antibody. In another preferred embodiment, the antibody is capable of inhibiting the interaction of a microorganism with a mammalian cell, preferably where the microorganism is a bacteria or yeast, and more preferably where the microorganism is C. albicans. In one other preferred embodiment of the antibody, the mammalian cell is a human cell, preferably an epithelial cell, more preferably a mucosal epithelial cell, and most preferably a buccal epithelial cell.
Another aspect of the invention is a method of preventing or treating infection by a microorganism of a mammalian host comprising the steps of administering to said host an effective amount of purified Hwp1 protein, antibody against said Hwp1 protein, or polypeptide comprising the amino acid sequence of SEQ. ID NO. 1, wherein said polypeptide is capable of acting as a substrate for mammalian transglutaminases, antibody against said polypeptide, purified proline-rich protein, or antibody against said proline-rich protein, in a pharmaceutically acceptable sterile vehicle, and inhibiting the interaction of said microorganism with the cells of said host. In another preferred embodiment, the antibody is capable of inhibiting the interaction of a microorganism with a mammalian cell, preferably where the microorganism is a bacteria or yeast, and more preferably where the microorganism is C. albicans. In one other preferred embodiment, the mammalian cell is a human cell, preferably an epithelial cell, more preferably a mucosal epithelial cell, and most preferably a buccal epithelial cell. In yet another preferred embodiment, the administering is performed orally. In a preferred embodiment, the mammalian host is immunocompromised, and in another, the infection is associated with AIDS.
Another aspect of the invention is a vaccine for preventing infection by a microorganism of a mammalian host comprising an effective amount of purified Hwp1 protein, antibody against said Hwp1 protein, or polypeptide comprising the amino acid sequence of SEQ. ID NO. 1, wherein said polypeptide is capable of acting as a substrate for mammalian transglutaminases, antibody against said polypeptide, purified proline-rich protein, or antibody against said proline-rich protein, in a pharmaceutically acceptable sterile vehicle, wherein said vaccine is capable of inhibiting the interaction of said microorganism with the cells of said host. In another preferred embodiment, the vaccine is capable of inhibiting the interaction of a microorganism with a mammalian cell, preferably where the microorganism is a bacteria or yeast, and more preferably where the microorganism is C. albicans. In one other preferred embodiment, the mammalian cell is a human cell, preferably an epithelial cell, more preferably a mucosal epithelial cell, and most preferably a buccal epithelial cell. In yet another preferred embodiment, the administering is performed orally. In a preferred embodiment, the mammalian host is immunocompromised, and in another, the infection is associated with AIDS.
An aspect of the present invention is a diagnostic kit for detecting the presence or absence of a microorganism expressing a protein capable of acting as a substrate for mammalian transglutaminases, comprising an antibody against a polypeptide comprising the amino acid sequence of SEQ. ID NO. 1 (or an antigenic portion thereof), wherein said polypeptide (or an antigenic portion thereof) is capable of acting as a substrate for mammalian transglutaminases. In a preferred embodiment, the diagnostic kit further comprises a detectable label selected from the group consisting of calorimetric, enzymatic, fluorescent and radioactive labels. In another preferred embodiment, the microorganism is a bacteria or yeast, preferably a yeast, and more preferably C. albicans. 
Another aspect of the present invention is a method for detecting a microorganism expressing a protein capable of acting as a substrate for mammalian transglutaminases, comprising the steps of contacting a sample with an antibody against a polypeptide comprising the amino acid sequence of SEQ. ID NO. 1 (or an antigenic portion thereof), wherein said polypeptide (or an antigenic portion thereof) is capable of acting as a substrate for mammalian transglutaminases, and detecting any binding of said microorganism with said antibody. In a preferred embodiment, the antibody is immobilized to a solid support. In another preferred embodiment, the antibody is conjugated to a detectable label selected from the group consisting of colorimetric, enzymatic, fluorescent and radioactive labels.
Yet another aspect of the invention is a method of preventing or treating infection by a microorganism of a mammalian host, comprising the steps of administering syngeneic host cells transformed with the vector comprising DNA encoding the polypeptide of SEQ. ID NO. 1, wherein said transformed syngeneic host cells produce a protein capable of acting as a substrate for mammalian transglutaminases and inhibiting the interaction of said microorganism with the cells of said host. In a preferred embodiment, the transformed syngeneic host cells are capable of inhibiting the interaction of a microorganism with a mammalian cell, preferably where the microorganism is a bacteria or yeast, and more preferably where the microorganism is C. albicans. In one other preferred embodiment, the mammalian cell is a human cell, preferably an epithelial cell, more preferably a mucosal epithelial cell, and most preferably a buccal epithelial cell. In yet another preferred embodiment, the administering is performed orally. In a preferred embodiment, the mammalian host is immunocompromised, and in another, the infection is associated with AIDS.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and the specific examples, however, indicate only preferred embodiments of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.